This research program addresses how a neuropeptide modulates olfactory processing of volatile pheromone information and shapes downstream courtship behavior. We are approaching this issue as a Multi-PI team using the model genetic system, Drosophila. The CG4395 gene encodes a Class B peptide G protein-coupled receptor (GPCR) highly related to mammalian receptors for Calcitonin and Calcitonin Gene Related Peptide. CG4395 specific antibodies and CG4395-promoter constructs reveal that CG4395 is prominently expressed in olfactory receptor neurons that respond to pheromone as well as in higher regions of the brain that have been implicated in courtship behavior and plasticity. Significantly, mutant male flies that lack CG4395 gene expression exhibit aberrant courtship behavior. Furthermore, this behavioral defect can be restored to mutant animals by expressing a CG4395 transgene in olfactory sensory neurons. These findings lead us to hypothesize that peptide neuromodulation via CG4395 regulates courtship-relevant pheromone processing. The CG4395 GPCR is an orphan, but it responds potently to a peptide factor in Drosophila head extracts which we have greatly enriched by three HPLC steps and now propose to purify to homogeneity. We also propose to use the power afforded by Drosophila genetics to study the CG4395 receptor and peptide ligand, separately and together, in vivo to learn where and when receptor signaling occurs. We will combine a battery of molecular genetic tools with detailed behavioral analyses to understand the roles of the receptor and ligand in courtship. In addition we will employ a novel genetically-encoded real-time reporter of cAMP levels that uses in vivo FRET measurement regulated by CG4395 as revealed by a novel genetic reporter. To achieve these goals demands skills greater than can be assembled in any current Drosophila laboratory. Therefore, this research program represents the collaborative efforts of three independent groups, each of which contributes specific expertise and technical experience. Neuromodulatory transmitter systems regulate numerous higher brain functions including attention, memory, mood, appetite, social behavior and aggression. When transmitter systems go awry, clinical problems often arise. Identifying modulatory peptides and cognate receptors is therefore a key endeavor in studying the mechanisms underlying behavior. GPCRs and their signaling pathways are highly conserved in evolution, thus the molecular mechanisms of neuromodulation we aim to describe will offer potential avenues for therapeutic intervention. PUBLIC HEALTH RELEVANCE Neuromodulation is fundamentally important for higher brain functions and regulates diverse brain-based phenomena including attention, memory, mood, appetite and aggressive behaviors. The normal actions of monoamine and peptide transmitters systems are keys to such modulation and when these transmitter systems go awry, these processes are compromised and clinical problems arise. This research program uses biochemical and genetic approaches to explore fundamental mechanisms underlying modulation of the neural circuits that regulate reproductive behaviors (courtship) in the model system Drosophila.